ESI-MS/MS quantification of 7α-hydroxy-4-cholesten-3-one facilitates rapid, convenient diagnostic testing for cerebrotendinous xanthomatosis

Background: The genetic disorder cerebrotendinous xanthomatosis (CTX) frequently remains undiagnosed for many years. Left untreated CTX is associated with the development of cataracts, xanthomas and severe neurological dysfunction. The method routinely used to screen for CTX is GC-based measurement of elevated 5α-cholestanol from hydrolyzed plasma. A plasma test for CTX utilizing ESI-MS/MS methodology would be beneficial. Methods: Development of rapid, simple LC-ESI-MS/MS methodology to test plasma for CTX is described. Two hour Girard derivatization allowed for 7α-hydroxy-4-cholesten-3-one quantification by isotope dilution LC-ESI-MS/MS within 12 min from un-hydrolyzed affected patient plasma (5 μl). Results: Adequate sensitivity and reproducibility were achieved for quantification of 7α-hydroxy-4-cholesten-3-one, which demonstrated improved utility as a diagnostic marker of disease and to monitor treatment compared to 5α-cholestanol. The mean plasma concentration of 7α-hydroxy-4-cholesten-3-one in untreated CTX-affected patients (n = 6) was 107-fold that in unaffected subjects (n = 9), with the lowest concentration in affected patients > 10-fold the highest concentration in unaffected subjects. Conclusion: Quantification of the bile acid precursor 7α-hydroxy-4-cholesten-3-one with LC-ESI-MS/MS is a novel approach to improved diagnostic testing of plasma for CTX, amenable to high-throughput analysis and automated sample handling. Development of ESI-MS/MS methodology should make CTX testing more widely available and facilitate easier diagnosis of CTX.